Abstract Balkan endemic nephropathy (BEN) is a degenerative disease that is often associated with urothelial cancer and is geographically confined to rural parts of Serbia, Bulgaria, Romania, Croatia, and Bosnia. The etiology of BEN is debated, and several environmental factors have been considered as contributors to the disease. The Pliocene lignite hypothesis links BEN and exposure to organic material in drinking water that is leached from a low-rank coal known as lignite. This hypothesis addresses the limited geographic nature of BEN, as most of the villages plagued with the disease are also closely associated with lignite deposits. We and others have shown an association of lignite deposits in the southeastern US with renal cell toxicity in culture and with an increased incidence of end-stage renal disease (ESRD).The overall goal of the studies in this work are to test the specific hypothesis that environmental levels of U.S. Gulf Coast region lignite aqueous extracts in drinking water result in BEN-like nephrotoxicity in vivo. Aim 1 will test the prediction that drinking water exposure to lignite coal extracts from the southeastern US results in a dose-dependent nephrotoxicity. In this study, male and female Sprague-Dawley (S-D) rats will be exposed to environmental levels of coal extracts in their drinking water for six months. Samples will be either lignite extracts found to be toxic to renal cells (Dolet Hills, LA and Monticello, TX); or lignite extracts found to be less toxic to kidney cells (San Miguel, TX; Hot Springs County, AR). Water intake, animal weights and general health will be assessed weekly. Blood and 24 hr urine will be collected at 3 and 6 months from a subset of animals and assessed for renal function markers (urine osmolality, NAG levels; serum urea nitrogen and creatinine). Kidneys will be assessed for toxicity, cell death and inflammation grossly and microscopically by a veterinary pathologist as will other potential target organs (bladder, lungs, heart, liver, spleen, brain, skin, complete blood count). Aim 2 will test the prediction that lignite degradation products, specifically polar organic compounds, are cytotoxic to renal cells. In this aim, toxic extracts (Dolet Hills, LA; Monticello, TX) will be separated by solid-phase extraction followed by a two-step elution process to generate a non-polar component (compounds soluble in hexane) and polar component (compounds soluble in methanol). These fractions applied at various concentrations as in our preliminary work to HK-2 and RPTEC/TERT1 renal cells and assessed for cytotoxicity. The impact of this proposal is to begin to tie these two findings together, namely to test whether lignite coal extracts in the drinking water of rats is nephrotoxic and to begin to understand the lignite components/mechanisms mediating this nephrotoxicity.